DESCRIPTION: The applicant's study of the cell cycle regulatory protein FP21 in the cellular slime mold Dictyostelium discoideum has led to the discovery of a novel pentasaccharide attached to a hydroxylated Pro-residue at position 143. This is a bulky substituent to be located on a highly conserved surface of a protein. It is also an unusual structure to find on a cytoplasmic/nuclear protein, as Pro hydroxylation and complex glycosylation are usually thought to be confined to the secretory pathway of the cell. The applicant's long term goals are to understand how this complex postranslational covalent modification, which cannot be predicted from cDNA sequence data, affects the function of FP21 and other proteins on which it presumably occurs. This project will focus on how the pentasaccharide is biosynthesized, initially using FP21 as a substrate to identify potentially key enzymes in the pathway. This will involve first establishing the specificity of the enzymes which govern its biosynthesis by completing the structure determination of the HyPro pentasaccharide (aim #1). This will be followed by purification of 2 early enzyme activities of the pathway, the ProHydroxylase and the GalNAcTransferase, and examining their substrate specificity and kinetic constants (aim#2). This approach is expected to identify candidate enzymes for the pathway, suggest the cellular compartment in which each operates, and the basis for its substrate recognition. Cloning cDNAs for these 2 enzymes and the cFucTase (aim#3) will lead to the primary structure of each enzyme protein, which will independently address their compartmentalization, suggest an evolutionary relationship with corresponding known enzymes in the secretory pathway, and provide tools for critical manipulations in the later aims. The sufficiency of each enzyme polypeptide for catalyzing its proposed step of the pathway will be established by expression of its cDNA in a heterologous system followed by enzymatic assay (aim #4). Finally, the necessity of the enzyme polypeptide for execution of the pathway step in vivo, and its co-compartmentalization with FP21, will be addressed by genetic disruption of the enzyme genes and expression of FP21 in the secretory pathway, respectively in Dictyostelium (aim#5). In addition to firmly establishing the basic principles by which the HyPro pathway initially targets and modifies FP21, the results will provide an important gateway through which we can detect this pathway in other organisms, regardless of whether FP21 is glycosylated in the particular cell or tissue type examined. The tools developed will permit future dissection of the role of complex glycosylation of cytoplasmic/nuclear proteins which may, based on the phenotype of FP21 and glycosylation mutants, be fundamentally important for cell proliferation and metabolism.